Of the present invention open
Once from octopus, separating in the trial of GnRH, by studying peptide in the activity aspect the heartbeat of enhancing animal, inventors of the present invention have carried out broad research to multiple physiologically active peptide, and these physiologically active peptides are brain samples of taking from about l00 kind 0.Vulgaris.As a result, inventors of the present invention separate and purifying have the peptide of following amino acid sequences (1), determined its structure, and by synthetic fully its structure of having verified:
<Glu-Asn-Tyr-His-Phe-Ser-Asn-Gly-Trp-His-Pro-Gly-NH
2?(1)
Wherein<Glu represents Pyrrolidonecarboxylic acid.This peptide is called compound (1) hereinafter.Unless otherwise indicated, all amino acid are by the trigram coded representation of IUPAC naming system definition.
As shown in Figure 5, compound (1) has very high homology with the GnRH that identifies in vertebrates.Different with known decapeptide, proved that compound (1) is a dodecapeptide.
Inventors of the present invention further utilize quail to test, and prove that compound (1) has the GnRH activity, also, cause the activity of lutropin (LH) from the pituitary gland release of quail.
Inventors of the present invention have also prepared primer from above-mentioned aminoacid sequence, and on the total RNA that from the brain of 0.Vulgaris, obtains, utilize ThermoScript II polymerase chain reaction (RT-PCR) to carry out gene sequencing.Inventors of the present invention have determined that by this way propeptide polypeptide of the present invention has the full length amino acid primary sequence shown in SEQ ID NO:2.
Inventors of the present invention have determined that further the gene of coding precursor polypeptide has the base sequence shown in the SEQ IDNO:3, thereby have finished the present invention.
In addition, two amino-acid residue Asn-Tyr are inserted in N-terminal zone by the decapeptide GnRH between first<Glu and second His, inventors of the present invention utilize the decapeptide GnRH that identifies from vertebrates to synthesize a kind of new peptide and have checked its physiologically active, and this peptide is with isolating from the brain of 0.Vulgaris and to have a peptide of aminoacid sequence (1) similar.
In other GnRH, have been found that chicken II type GnRH (cGnRH-II) shown in Figure 5 is prevalent in chicken and other multiple animals.Therefore, the N-terminal zone with two amino-acid residue Asn-Tyr insert the cGnRH-II between first<Glu of peptide section and second His residue has obtained the represented new peptide of following amino acid sequences (2):
<Glu-Asn-Tyr-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH
2?(2)
Wherein<Glu represents Pyrrolidonecarboxylic acid.This peptide is called compound (2) hereinafter.The activity of detection compound (2) aspect enhancing 0.Vulgaris heartbeat, we observe compound (2) and show and the similar activity of isolated compound (1) from the brain of 0.Vulgaris, but cGnRH-II does not show this activity.
In addition, inventors of the present invention make be separated to from invertebrates and have 2 and 3 amino acids in the active decapeptide of GnRH disappearance N-terminal zone, thereby be translated into a kind of and the similar decapeptide of isolating GnRH from vertebrates.As a result, we observe this decapeptide and have kept initial GnRH activity.
In order to redescribe, inventors of the present invention have determined that the represented peptide of following amino acid sequences (3) has the GnRH activity, and this peptide is to obtain by amino acid 2 and 3 is removed in the N-terminal zone of isolated compound (1) from the brain of 0.Vulgaris:
<Glu-His-Phe-Ser-Asn-Gly-Trp-His-Pro-Gly-NH
2(3). wherein<Glu represents Pyrrolidonecarboxylic acid.This peptide is called compound (3) hereinafter.
Accordingly, the invention provides and have gonadotropin-releasing hormone (GnRH) is active and have the decapeptide of following amino acid sequences (I):
<Glu-Zaa-Zaa-His-Zaa-Ser-Zaa-Zaa-Zaa-Zaa-Pro-Gly-NH
2?(I)
Wherein<Glu represents Pyrrolidonecarboxylic acid, and Zaa represents any one amino acid.
Can limit more specifically peptide above-described, that have the active aminoacid sequence of GnRH (I) by following amino acid sequences (II):
<Glu-Zaa-Zaa-His-Zaa-Ser-Zaa-Gly-Zaa-Zaa-Pro-Gly-NH
2?(II)
Wherein<Glu represents Pyrrolidonecarboxylic acid, and Zaa represents any one amino acid.
Can limit more specifically peptide above-described, that have the active aminoacid sequence of GnRH (I) by following amino acid sequences (III):
<Glu-Zaa-Zaa-His-Yaa-Ser-Zaa-Gly-Zaa-Zaa-Pro-Gly-NH
2?(III)
Wherein<and Glu represents Pyrrolidonecarboxylic acid, and Zaa represents any one amino acid, and Yaa represents die aromatischen Aminosaeuren.
Provided by the invention have in the active peptide of GnRH, and especially preferred is those by following amino acid sequences (1), (2), (3) represented peptide:
<Glu-Asn-Tyr-His-Phe-Ser-Asn-Gly-Trp-His-Pro-Gly-NH
2?(1)
<Glu-Asn-Tyr-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH
2?(2)
<Glu-His-Phe-Ser-Asn-Gly-Trp-His-Pro-Gly-NH
2?(3)。
Wherein<Glu represents Pyrrolidonecarboxylic acid.
The present invention further provides method of producing pharmaceutical preparation or sterilant and the method for producing young octopus, had an active aminoacid sequence of GnRH (I) and be used as activeconstituents and contain in pharmaceutical preparation or the sterilant.
According to one aspect of the present invention, the invention provides the precursor polypeptide, thereby and these precursor polypeptide contain the represented aminoacid sequence of SEQ ID NO:2, contain by the represented aminoacid sequence of SEQ ID NO:2 being carried out part is removed or replacing the aminoacid sequence that obtains or contain at those by the represented aminoacid sequence of SEQ ID NO:2 being carried out part and remove or replacing the sequence that interpolation 1 on the aminoacid sequence that obtains or a plurality of amino acid obtain.
According to another aspect of the present invention, the invention provides the peptide in the code book invention and the gene of precursor polypeptide thereof, gene, the carrier that has mixed these genes with the represented base sequence of SEQ ID NO:3 especially is provided, utilizes the host that these carriers transformed and utilized these genes and prepare the peptide among the present invention or the method for precursor polypeptide by genetically engineered.
Implement best mode of the present invention
Novel peptide provided by the invention can be done in order to cause that lutropin (LH) discharges and quicken the heartbeat of 0ctopus vulgaris from the quail pituitary gland.Therefore, on its active basis,, can from the brain of 0.vulgaris, separate and these peptides of purifying according to the following step to the 0.vulgaris heart:
In one embodiment, the hot water extract who has prepared the 0.vulgaris brain.Be added with 3% acetate in the extracting solution.With the extracting solution cooling, carry out centrifugal subsequently to obtain crude extract.Extract is adsorbed onto (for example, WATERS Co., the Ltd production of a C18 column extractor
Vac C18 column extractor) on, utilize 60% methyl alcohol that contains 0.1% trifluoroacetic acid to carry out wash-out subsequently to collect peptide moiety.Peptide moiety is carried out ion exchange chromatography, reversed phase chromatography or other chromatographic technique separates and purifying purpose peptide.
As a decapeptide, utilize by common peptide synthesizer (as PE biosystem JapanCo., the peptide synthesizer 433A that the Ltd. makes) solid-phase synthesis that carries out or other ordinary skills in the synthetic organic chemistry and just can synthesize peptide among the present invention easily.In the time of necessary, utilize thick peptide prod that these technology obtain to carry out purifying by common purification technique such as RPLC and crystallization.
Utilize following method can determine the precursor amino acid sequence of polypeptide of peptide section and the base sequence of the precursor polypeptide in the invention of definite code book:
Synthetic primer on the aminoacid sequence basis of the peptide that utilization obtains with aforesaid method carries out ThermoScript II polymerase chain reaction (RT-PCR) to the total RNA for preparing from the brain of 0.vulgaris, determine that gene order is about 750bp.Subsequently, utilize 5 '-RACE and 3 '-RACE technology determines respectively 5 ' and 3 ' terminal gene order. according to the present invention, determined that propeptide polypeptide of the present invention contains the aminoacid sequence shown in the SEQ ID NO:2, and the gene of this precursor polypeptide of having determined to encode contains the base sequence shown in the SEQ ID NO:3.
Therefore, can utilize recombinant DNA technology to prepare propeptide polypeptide among the present invention and the peptide among the present invention. when wanting to utilize recombinant DNA technology to prepare the precursor polypeptide, the carrier that has mixed the gene shown in SEQ ID NO:3 can be imported among the host and transform.Subsequently, cultivate the host or make its growth, from host or host's nutrient solution, separate and purifying purpose precursor polypeptide.
In order from the precursor polypeptide, to obtain the purpose peptide, can utilize enzyme that the precursor polypeptide is processed and modified.If necessary, can carry out isolated or purified to peptide prod.
Each peptide among the present invention all has the activity that strengthens the 0.vulgaris heartbeat and has the closely similar structure with vertebrate GnRH (peptide).These peptides also act on the pituitary gland of quail and cause that lutropin discharges.In addition, to studies show that the peptide among the present invention carries out, bird GnRH is relevant with the activity that strengthens the octopus heartbeat.These observations show jointly, the peptide among the present invention be actually with octopus and other invertebratess and vertebrates in the relevant hormone of release of gonadotropin.
Therefore, the peptide among the present invention can be used as octopus biology and the GnRH of research invertebrates and the useful materials of the structural relation between vertebrate GnRH that research comprises the reproduction mechanism of octopus.Therefore, these peptides have important effect in evolving zoologizeing. in addition, we wish that not only the peptide among the present invention has in the active novel substance of gonadotropin-releasing hormone in exploitation and has use, and wish that they play a role at the new technology aspect that utilizes aspect peptide itself or its analogue developing new drug and the sterilant and produce young octopus.
For example, the peptide among the present invention can be used as active ingredient of drugs, and acceptable vehicle is mixed with suitable oral and parenteral formulations, for example capsule, tablet and injection on pharmacology.Especially, the peptide among the present invention can mix with vehicle such as lactose, starch or their derivative or with derivatived cellulose, and wraps into and form capsule preparations in the gelatine capsule.
When we wanted that the peptide among the present invention is prepared into tablet, the vehicle that will be mentioned before added tackiness agent such as Xylo-Mucine, Lalgine and Sudan Gum-arabic and water outward.Subsequently this mixture is carried out kneading, and if necessary, just be made into particle.Subsequently, in mixture, add lubricant such as talcum powder and Magnesium Stearate and utilize common compressed tablets draft machine that it is prepared into tablet.
When we want that the peptide among the present invention is prepared into the preparation that is used for administered parenterally, be dissolved in sterile distilled water or Sterile Saline with solubilizing agent peptide and the ampoule that places sealing to be provided for the preparation of drug administration by injection.In the time of necessary, stablizer, buffer reagent or other additives can be added in the preparation.In addition, the peptide among the present invention can leave in the phial by powder type, like this, thereby just powder dissolution can be formed pharmaceutical solutions in sterile distilled water when using.These parenteral administrations can be sent by intravenously administrable or by intravenous drip.
Preferably,, comprise disease to be treated, patient's symptom, severity, patient's age and the route of administration of symptom, adjust containing as the dosage of the preparation of the peptide of the present invention of activeconstituents in view of multiple factor.When carrying out oral administration, the dosage of said preparation is generally 0.1-l000mg/ days/people, preferred 1-500mg/ days/people, and when carrying out administered parenterally, the dosage of said preparation can be about the 1/100-1/2 of oral administration.
In view of its GnRH essence, the peptide among the present invention can give the Cephalopoda animal, thereby makes animal just reach sexual maturity in early days, but so just induced animal begins to lay eggs in early days.Breed and raise these animal capable faster production young Cephalopoda animals.
Embodiment
Hereinafter will be described in detail the present invention, but this does not limit the intention of invention scope of the present invention by any way by embodiment.
Embodiment 1: centrifugation is in the peptide section of heart from 0.vulgaris
(a) the thick extraction
From 100 0.vulgaris samples, take out cerebral tissue (comprising optic gland, tractus opticus and optic lobe), freezing in liquid nitrogen.In 900ml distilled water, refrigerated tissue (180g) was boiled 10 minutes.After the cooling, add 3% acetate, with tissue homogenate and under 4 ℃, 12,000 * g centrifugal 30 minutes to obtain supernatant liquor.In pellet, add the acetate of 200ml3%, to mixture once more homogenate for use in extracting.Subsequently, under the same conditions mixture is carried out centrifugal to obtain supernatant liquor.Collect supernatant liquor and vacuum concentration to about 200ml so that crude extract to be provided.
(b) be adsorbed onto on the C18 extraction cartridge
In the crude extract that in step (a), obtains, add 5ml6MHCl, with this mixture at 4 ℃, 12, under the 000g centrifugal 30 minutes.The supernatant liquor that obtains is passed through Sep-
VacC18 extraction cartridge (10g, 35cc, WATERS Co., Ltd. produce) so that solute wherein be adsorbed onto on the extraction cartridge. subsequently, (TFA hereinafter) washes, and utilizes 100ml 60% methyl alcohol/0.1%TFA wash-out to be retained in the material on the post to utilize 200ml 0.1% trifluoroacetic acid.Eluate is carried out vacuum concentration and lyophilize, obtain 150mg dry labor thing.
(c) reversed phase high efficiency liquid phase column chromatography (1)
Utilize Capcell pak C18 UG80 (5 μ m, 10mm x 250mm, SHISEIDOCo., Ltd. manufacturing) that the dry labor thing that obtains in the above-mentioned steps (b) is carried out reversed phase high efficiency liquid phase column chromatography (rp-hplc).Especially, the dry labor thing that is dissolved among the 0.1%TFA (pH2.2) is crossed post with the speed of 1.5ml/min, utilizes acetonitrile linear gradient elution material retained, and be dissolved in acetonitrile concentration among the TFA in 60 minutes from 0% linear change to 60%.On the basis of 215nm uv-absorbing, elutriant is carried out the fraction collection of 3ml.
According to the scheme of describing among the embodiment 5 each part is carried out biological assay, found that, the part that elutes when acetonitrile concentration changes between 26%-30% has the activity that strengthens the 0.vulgaris heartbeat.
(d) cation exchange column chromatography
Utilize tsk gel SP-5PW (10 μ m, 7.5mm x 75mm, TOSOH Co., Ltd. manufacturing) that the active part that obtains in the above-mentioned steps (c) is carried out cation exchange column chromatography (cationic exchange HPLC).Particularly, join this active part in the post and utilize the flow velocity wash-out of 10mM phosphoric acid buffer (pH7.0) with 1.0ml/min, wherein the NaCl concentration in the phosphoric acid buffer was carried out linear change (linear gradient) from 0.1M to 0.6M in 60 minutes.Elutriant is carried out the fraction collection of 2ml and each collection part is carried out biological assay.The wash-out of collecting during for 0M when the NaCl concentration in the damping fluid partly has activity.
(e) reversed-phase HPLC (2)
Utilize Capcell pak C18 UG80 (5 μ m, 4.6mm x 150mm, SHISEIDOCo., Ltd. manufacturing) that the active part that obtains in the above-mentioned steps (d) is carried out reversed-phase HPLC.Particularly, join this active part in the post and utilize 0.1%TFA (pH2.2) to carry out wash-out with the flow velocity of 1.0ml/min, wherein the acetonitrile concentration among the TFA carried out linear change (linear gradient) from l0% to 30% in 60 minutes.Elutriant is carried out the fraction collection of 1ml.The part that wash-out goes out when the concentration of acetonitrile is about 19.5% has activity.
(f) reversed-phase HPLC (3)
Utilize Capcell pak C18 UG80 (5 μ m, 4.6mm x 150mm, SHISEIDOCo., Ltd. manufacturing) that the active part that obtains in the above-mentioned steps (e) is carried out reversed-phase HPLC.Particularly, this part is joined in the post, utilize the 0.1%TFA (pH2.2) contain 18% acetonitrile to carry out wash-out with the flow velocity of 0.5ml/min.When retention time is 17.5 minutes, obtain one and shown unimodal compound.With this compound called after compound (1).Fig. 1 has showed the result of reversed-phase HPLC.
Embodiment 2: the structure of compound (1) is determined
Because the N-terminal of modified exists, the aminoacid sequence of peptide is can not determine in the structural analysis that the compound (1) that utilizes Shimadzu PSQ-1 protein sequencing instrument (SHIMADZU Vo., Ltd. makes) that purifying among the embodiment 1 is obtained carries out.Therefore, the composition of the amino acid in the peptide is analyzed.
Table 1: the amino acid of peptide is formed
Amino acid |
pmol |
Mol ratio |
Asx |
51.1 |
1.5 |
Glx |
42.6 |
1.3 |
Ser |
32.7 |
1.0 |
Gly |
70.0 |
2.1 |
His |
57.9 |
1.7 |
Pro |
37.1 |
1.1 |
Tyr |
24.2 |
0.7 |
Phe |
33.8 |
1.0 |
(Micromass, UK Co. Ltd.) determine the molecular weight of compound (1) by Q-TOF.The data that record are as shown in table 2 below.
Table 2: the mass-spectrometric data of peptide
Compound |
Molecular formula |
Calculated value (M+2H)
2+ |
Calculated value (M+2H)
2+ |
Before enzyme is handled |
C
66H
80N
20O
17 |
713.30 |
713.30 |
After enzyme is handled |
C
61H
75N
19O
15 |
657.79 |
657.85 |
Can determine that from amino acid composition analysis and molecular weight determination the N-terminal of compound (1) contains a Pyrrolidonecarboxylic acid.Accordingly, utilization can be excised enzyme (Ltd. produces for Pyrrolidonecarboxylic acid aminopeptidase, TAKARA SHUZO Co.) the processing compound (1) of first Pyrrolidonecarboxylic acid residue and be utilized the protein sequencing instrument to analyze once more.
As a result, determined the aminoacid sequence of compound (1) since second amino-acid residue, and the alleged occurrence Trp that can not detect by amino acid composition analysis.
The aminoacid sequence of determining is as shown in the table.
Table 3: the aminoacid sequence of the peptide after enzyme (pmol) is handled
Circulation |
Amino acid |
1 |
Asn?27 |
2 |
Tyr?25 |
3 |
His?7 |
4 |
Phe?23 |
5 |
Ser?3 |
6 |
Asn?16 |
7 |
Gly?12 |
8 |
Trp?3 |
9 |
His?2 |
10 |
Pro?5 |
11 |
Gly?4 |
In order to guarantee that enzyme is handled is to act on according to desired, in the same way, utilize Q-TOF (Micromass, UK Co., Ltd.) molecular weight to the compound (1) after the enzyme processing redeterminates, and the result confirms to have excised first Pyrrolidonecarboxylic acid residue.The result is also as shown in table 2.
By aforesaid instrumental analysis, we have determined compound (1), promptly separate from 0.vulgaris and the neuropeptide of purifying, have following aminoacid sequence (1):
<Glu-Asn-Tyr-His-Phe-Ser-Asn-Gly-Trp-His-Pro-Gly-NH
2?(1)
Wherein<Glu represents Pyrrolidonecarboxylic acid.
Embodiment 3: by solid phase synthesis process synthetic compound (1)
According to
Chemistry utilizes fully automated peptide synthesizer 433A (PEBiosystems Japan) synthetic compound (1).
In synthetic, utilize Fmoc-NH-SAL-A resin (WATANABE CHEMICALINDUSTRIES, Ltd. make) as solid support, and use Pyrrolidonecarboxylic acid, Fmoc-Asn-(Trt), Fmoc-Tyr-(tBu), Fmoc-His-(Trt), Fmoc-Phe, Fmoc-Ser-(tBu), Fmoc-Gly, Fmoc-Trp-(tBoc) and Fmoc-Pro simultaneously.
As used herein, Fmoc represents 9-fluorenyl methoxy carbonyl, and tBu represents the tertiary butyl, and Trt represents trityl, and tBoc represents the t-tert-butoxycarbonyl.
Building-up reactions finish and to thick peptide go the protection after, utilize 10ml contain the 100mg 2 methyl indole 2.5% 1, the mixture of 2-ethane two mercaptan/2.5% water/95%TFA cuts away thick peptide from peptide resin.After reaction is finished, reaction mixture is filtered. in filtered liquid, add ether so that the peptide precipitation.Utilize ether to precipitation flushing 3 times, obtain the thick peptide of about 100mg, subsequently the thick peptide of about 10mg is carried out purifying by reversed-phase HPLC, obtain the peptide of about 6mg purifying.
When utilizing Capcell pak C18 UG80 to carry out reversed-phase HPLC, the peptide of purifying is coming out with the identical retention time wash-out of compound (1).In addition, the synthetic peptide is showing the activity identical with native peptides aspect the heartbeat that strengthens 0.vulgaris.
Embodiment 4: by solid phase synthesis process synthetic compound (2) and (3)
Utilize with embodiment 3 above in identical method, synthesized following amino acid sequences (2) and (3) represented peptide:
<Glu-Asn-Tyr-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH
2?(2)
<Glu-His-Phe-Ser-Asn-Gly-Trp-His-Pro-Gly-NH
2?(3)。
Wherein<Glu represents Pyrrolidonecarboxylic acid.
Embodiment 5: the heartbeat activity of measuring 0.vulgaris
Method according to propositions such as Morishita is measured (Fumihiro Morishita, Biochem.Biophys.Res.Commun., 240,354-358 (1997)) to 0.vulgaris heartbeat activity.Particularly, take out the heart of 0.vulgaris, and from heart, take out the atrium.From each atrium, insert intubate in ventricle, and utilize cotton thread to tie up simultaneously intubate is connected in the chamber (volume is 80ml). aorta is clamped in order to avoid liquid is revealed from ventricle, be connected with a pressure transmitter then.Heart after being ready to complete is used as working sample. adjust every conduit to the speed that allows synthetic sea water (containing 0.1% glucose) with 1-2ml/min and flow.Each analysans is dissolved in the identical synthetic sea water of 1ml, and this solution is expelled in the heart by intubate.The variation of record heartbeat.
Choose isolated compound from the brain of 0.vulgaris (1), compound (2) and cGnRH-II, vertebrates GnRH that from chicken, be separated to and that be used for derivative compound (2) as testing compound.
As shown in Figure 2, the result shows that compound (1) has strengthened the heartbeat of 0.vulgaris.
Shown in Fig. 3 (b), compound (2) has also strengthened the heartbeat of 0.vulgaris by raising heart rate and heartbeat amplitude.On the contrary, from as Fig. 3 (a) as can be seen, cGnRH-II does not influence heart rate and the heartbeat amplitude of 0.vulgaris.
Embodiment 6: to quail influence pituitary
Inoculation 7.54 x 10 in each hole of 24 orifice plates
5The individual quail cell pituitary (pituitary gland with two quails is suitable basically) of taking from.Subsequently, hatch these cells in containing the substratum of foetal calf serum, the time is 3 days.In 1ml does not contain the substratum of serum, these cells are not carried out extra 24 hours hatching.After hatching, respectively with 0,10
-9, 10
-8, 10
-7, 10
-6With 10
-5The final concentration of M adds peptide solution in substratum, and further these cultures is hatched 4 hours.From these cultures, collect supernatant liquor subsequently.On each concentration level, 5 samples are carried out radioimmunoassay and recording detection data.Utilize one-way analysis of variance (ANOVA) that data are carried out statistical study.
The result as shown in Figure 4.As shown in Figure 4, statistic analysis result shows, compound (1), and peptide promptly of the present invention has significantly improved the release of lutropin (LH).
Utilize identical method to detect, prove that compound (3) also can improve the release of lutropin (LH).
Embodiment 7: determine the complete amino acid structure of precursor polypeptide and determine the gene of coding this polypeptide
Base sequence
(1) total RNA of preparation 0.Vulgaris brain
In liquid nitrogen, the brain of about 1g0.vulgaris is pulverized, be dissolved in 10ml
Reagent (GIBCO BRL Co., Ltd) in and homogenate.Under the room temperature, homogenate was left
standstill 5 minutes and was packed as the equal portions of 1ml.In each equal portions, add 200 μ l chloroforms, and stir this mixture. subsequently, (SAKUMA SEISAKUSHO Co. Ltd.) carries out centrifugal (15,000rpm, 15min, 4 ℃) to this mixture to utilize refrigerated centrifuge.After centrifugal, the sucking-off upper aqueous layer is also added the 0.5ml Virahol to aqueous phase.Subsequently, at room temperature this mixture being left
standstill 10 minutes, utilize refrigerated centrifuge to carry out further centrifugal (15,000rpm, 10min, 4 ℃) then. the sucking-off supernatant liquor also will precipitate in the ethanol that is suspended in 1ml75% again.Suspension is carried out again centrifugal (10,000rpm, 5min, 4 ℃).Subsequently, remove supernatant liquor, will be deposited in air drying about 10 minutes.By under 60 ℃, hatching 10min the RNA flaky precipitate is dissolved in the 10 μ l DPEC treated waters.Utilize this method to obtain total RNA of about 3mg.
(2) 3 of degeneracy '-RACE
On the aminoacid sequence basis of isolating peptide from the brain of 0.vulgaris, design following degenerated primer and utilized usual way that they are synthesized:
Primer-1:5 '-CA (A/G) AA (C/T) TA (C/T) CA (C/T) TT (C/T) IIIAA (C/T) GG-3 '
(C/G) IAA (C/T) GGITGGCA (C/T) CCIGG-3 ' of primer-2:(T/A)
Wherein the foundation of each letter all is the definition (proving by experiment that to SHUJUNSHA Co., the cell engineering of Ltd. was replenished, and has hereinafter selected same code for use) of " nucleic acid code table ".
According to following rules, utilize 5 '/3 '-RACE test kit (Boehringer MannheimCo., Ltd.) carry out degeneracy 3 '-RACE: the synthetic damping fluid of the total RNA of 2 μ g, 4 μ l cDNA, 2 μ l dNTP mixtures, 1 μ l oligomerization dT anchor primer (12.5pmol/ μ l), 1 μ l AMV ThermoScript II (20 units/μ l) and DEPC treated water are added to together, make cumulative volume reach 20 μ l.Under 55 ℃, hatched this mixture 60 minutes, and under 65 ℃, hatched 10 minutes cDNA subsequently with synthetic article one chain.
Subsequently, according to following program carry out for the first time 3 '-RACE: with cDNA, 5 μ l, 10 x PCR damping fluids, 8 μ l dNTP mixtures, 5 μ l primers-1 (100pmol/ μ l), 1 μ l PCR anchor primer (12.5pmol/ μ l), the 0.5 μ l TaKaRaEx of 3 μ l article one chains
(TAKARA SHUZO Co., Ltd.) and water mix mutually, make cumulative volume reach 50 μ l.Hatched this
mixture 5 minutes under 94 ℃, and carried out 30 circulations subsequently, each circulation was included in 94 ℃ following 30 seconds, 49 ℃ following 30 seconds and 72 ℃ following 2 minutes, carried out 5 minutes hatch at 72 ℃ after 30 circulations.(Perkin ElmerCo. Ltd.) carries out the PCR reaction to select GeneAmp PCR System 2400 thermal cyclers for use.
Subsequently, utilize spin post (MicroSpin S-400, Amersham PharmaciaCo., Ltd makes) to the first time PCR product carry out purifying, and according to follow procedure carry out for the second time 3 '-RACE: with 5 μ l product, 5 μ l10 x PCR damping fluids, 8 μ l dNTP mixtures, 5 μ l primers-2 (100pmol/ μ l), 1 μ l PCR anchor primer (12.5pmol/ μ l), the 0.5 μ l TaKaRa Ex of PCR for the first time
(TAKARA SHUZOCo., Ltd.) and water mix mutually, make its cumulative volume reach 50 μ l.Mixture was hatched under 94
℃ 5 minutes, carry out 30 circulations subsequently, each circulation is included in 94 ℃ following 30 seconds, 51 ℃ following 30 seconds and 72 ℃ of following 1min, carries out hatching of 7min at 72 ℃ after 30 circulations.
Subsequently 5 μ l reaction mixtures are carried out electrophoresis on 1.5% sepharose.Observe and be about 400bp and about 600bp two bands, this shows that amplification has obtained two PCR products.
(3) collect the PCR product
5 μ l reaction mixtures are carried out electrophoresis and cut away two PCR product bands that form at about 400bp and about 600bp on 1.5% sepharose.(QIAGEN Co. Ltd) collects DNA from gel to utilize gel extraction kit.For the validating experiment result, respectively get the dna fragmentation that 3ml collects, on 1.5% sepharose, carry out electrophoresis.The result observes two PCR product bands that length is about 400bp and 600bp respectively.
(4) connect the PCR product
Utilize the spin post respectively the two PCR products that are about 400bp and 600bp to be carried out purifying.For each PCR product, PCR product, 2 μ l TA cloning vector pCR2.1 (Invitrogene Co. with 3 μ l, Ltd) and 5 μ l Ligation High (TOYOBO Co. Ltd) mutually mixes, and mixture is 16 ℃ of reactions 1 hour (ligation) down.
(5) colibacillary conversion
Respectively get two parts of bacillus coli DH 5 alpha (TOYOBO Co., Ltd) mixing that connect mixture and high energy that obtain in the 10 μ l steps (4).With mixture ice bath 30min, subsequently 42 ℃ of following heat shocks 50 seconds, then at cooled on ice 2min.After the cooling, to wherein adding the 1mlSOC substratum and under 37 ℃, hatching 10 minutes.Subsequently, 35 μ l X-gal are spread the LB/Amp nutrient agar surface of (LB contains the penbritin of 50ug/ml) uniformly, and 50 μ l transformant are inoculated so far on the substratum.Remaining transformant is 10, and centrifugal 1min makes its volume reach about 100 μ l under the 000rpm, then all transformant also is seeded in the LB/Amp nutrient agar.With this substratum 37 ℃ of following overnight incubation.
(6) bacterium colony PCR
The bacterium colony that utilizes previous step to obtain carries out bacterium colony PCR as template.Particularly, with Bacillus coli cells, 5 μ l 10x reaction buffers, 5 μ l 2mM dNTP, 3 μ l 25mM MgCl
2, 0.5 μ l M13FW primer (100pmol/ μ l), 0.5 μ l M13RV primer (100pmo l/ μ l), 0.5 μ l rTaq DNA polymerase (TOYOBO Co., Ltd) and water mix mutually, make its cumulative volume reach 50 μ l.Hatched this mixture 10 minutes under 90 ℃, and carried out 30 circulations subsequently, it is following 1 minute that each circulation is included in 94 ℃ following 30 seconds, 55 ℃ following 30 seconds and 72 ℃.3 μ l reaction mixtures are carried out electrophoresis on 1.5% sepharose.
Utilize synthetic M13FW of common method and M13RV primer, its base sequence is as follows:
M13FW:5′-GTAAAACGACGGCCAGTG-3′
M13RV:5′-GGAAACAGCTATGACCATG-3′
(7) dna sequence dna
Utilize the spin post bacterium colony PCR product of required size (about 600bp and about 800bp) to be carried out purifying and utilizes the dna sequence analysis test kit (PE biosystem Co. Ltd.) carries out sequential analysis to them.(PE biosystemCo. Ltd.) carries out sequential analysis to utilize ABI PRISM 310 genetic analysis instrument.
Utilize genetic analysis software GENETYX-MAC (SOFTWARE KAIHASTU Co.Ltd) that gene order is analyzed, determined to be about the cDNA sequence of 800bp.Further dna sequence analysis shows that the cDNA fragment that is about 600bp is actually the segmental part of the cDNA that is about 800bp.
(8)5′-RACE
Synthesized following primer according to the segmental base sequence of cDNA:
5′-RACE-1:5′-GGTCTGCAATCTCATTGACC-3′
5′-RACE-2:5′-TGCCAGTCAGGATTTCTCTC-3′
5′-RACE-3:5′-TCTTGGTGTTTGTCGGAAGG-3′
Utilize 5 '/3 '-RACE test kit (Boehringer Mannheim Co., Ltd.), with reference to following rules carry out 5 '-RACE: the total RNA of 2ug, 1 μ l 5 '-RACE-1 (12.5pmol/ μ l), the synthetic damping fluid of 4 μ l cDNA, 2 μ l dNTP mixtures, 1 μ l AMV ThermoScript II (20 units/μ l) and DEPC treated water are added to together, make cumulative volume reach 20 μ l.Under 55 ℃, hatched this mixture 60 minutes, and under 65 ℃, hatched other 10 minutes cDNA subsequently with synthetic article one chain.
After utilizing the spin column purification, the cDNA of article one chain is mixed with 2.5 μ l reaction buffers and 2.5 μ l 2mM dNTP, under 94 ℃, hatched this mixture 3 minutes, subsequently in cooled on ice.Subsequently, (10 units/μ l) add in this mixture with 1 μ l terminal enzyme (DNA), hatch this mixture 20 minutes under 37 ℃, hatch this mixture 10 minutes subsequently with the synthetic cDNA. that contains the dA tail under 70 ℃
Then, under following condition, carry out the first time and second time PCR:
(a) PCR for the first time
The cDNA, 5 μ l, 10 x PCR damping fluids, 8 μ l dNTP mixtures, 1 μ l 5 '-RACE-2 (12.5pmol/ μ l), 1 μ l oligomerization dT anchor primer (12.5pmol/ μ l), the 0.5 μ l TaKaRa Ex that 3 μ l are contained the dA tail
(TAKARA SHUZO Co., Ltd.) and water mix mutually, make cumulative volume reach 50 μ l. and under 94 ℃, hatched this
mixture 5 minutes, carry out 30 circulations subsequently, each circulation is included in 94 ℃ following 30 seconds, 55 ℃ following 30 seconds and 72 ℃ of following 3min, carries out hatching of 5min after 30 circulations under 72 ℃.
(b) PCR for the second time
With first time PCR product, 5 μ ls 10 x PCR damping fluids, 8 μ l dNTP mixtures, 1 μ l 5 '-RACE-3 (12.5pmol/ μ l), l μ l PCR anchor primer (12.5pmo l/ μ l), the 0.5 μ l TaKaRa Ex of 5 μ l behind the spin column purification
(TAKARASHUZO Co., Ltd.) and water mix mutually, make volume reach 50 μ l.Utilization is subsequently carried out PCR with the cycling condition that PCR is identical for the first time.
Utilize 1.5% sepharose to the second time PCR product carry out electrophoretic analysis.The result observes the band that is about 600bp.
According to above (4), (5), (6) and (7) middle rules of describing are determined the base sequence of PCR product for the second time.
By the analysis of front, determined size (about 700bp) and the base sequence of cDNA of the gene of the propeptide polypeptide in the code book invention.In addition, the amino acid estimated number (89 amino-acid residues) and the aminoacid sequence of the propeptide polypeptide among the present invention have been determined.
The base sequence of the gene (cDNA) of precursor amino acid sequence of polypeptide and coding precursor polypeptide is respectively shown in SEQ ID NO:2 and SEQ ID NO:3.In addition, Fig. 6 has showed the partial amino-acid series corresponding to the propeptide polypeptide among the present invention
In Fig. 6, mark with underscore corresponding to the aminoacid sequence of the peptide among the present invention.
Embodiment 8: demonstrative preparation
Tablet
Form:
Compound (1) 10g
Lactose 125g
Micro-crystalline cellulose 25g
W-Gum 25g
5% Vltra tears 100ml
Magnesium Stearate 5g
In accordance with known methods, mentioned component is carried out kneading, granulation, drying and typing to obtain tablet, every heavy 190mg also contains 100mg compound (1) as activeconstituents.
<120〉encoding gene of Xin Ying gonadotropin-releasing hormone, its precursor peptide and precursor peptide thereof
<223〉Xaa is the Pyrrolidonecarboxylic acid residue, also is<Glu.Its C-end has carried out amidation.